ABSTRACT: A method is presented to create microscale vortices using a microfluidic setup specifically designed to investigate the response of swimming microorganisms. Stable, small-scale vortices were generated in the side cavity of a microchannel by the shear stress in the main flow. The generation of a vortex was found to depend on the cavitys geometry, in particular its depth, aspect ratio, and opening length. Using video-microscopy, the position and orientation of individual bacteria and algae swimming in vortices of various strengths were tracked. Elongated cells were found to align with the streamlines in a strong vortex. The experimental system provides quantitative single-cell information on the interaction between microorganisms and their fluid dynamical environment.